External quality assurance of circulating tumor cell enumeration using the CellSearch(®) system: a feasibility study.
Author(s): Kraan J, Sleijfer S, Strijbos MH, Ignatiadis M, Peeters D, Pierga JY, Farace F, Riethdorf S, Fehm T, Zorzino L, Tibbe AG, Maestro M, Gisbert-Criado R, Denton G, de Bono JS, Dive C, Foekens JA, Gratama JW
Publication: Cytometry B Clin Cytom, 2011, Vol. 80, Page 112-8
PubMed ID: 21337491 PubMed Review Paper? No
Purpose of Paper
This paper evaluated how circulating tumor cell (CTC) counts are influenced by a pre-processing delay and inter-laboratory, intra-assay, inter-instrument, and inter-operator variability when enumerated using the CellSearch system.
Conclusion of Paper
The 14 participating laboratories reported similar CTC counts among peripheral blood aliquots that were subjected to a pre-processing delay of three days or less. Overall discordance among the 14 laboratories analyzing blood aliquots was 7.5% and inter-laboratory coefficients of variation (CV) ranged between 45-64%. Further, inter-instrument and inter-assay variation were both very low (CV<12% and CV=14-20%, respectively). Inter-operator agreement (based on Fleiss' K statistics) was classified as "substantial" or "almost perfect", but discordance for a given specimen ranged between 4 and 31%. The authors specified that scoring inconsistencies among laboratories was most prominent among samples that contained high numbers of cells that were dead, apoptotic, or lacked a nucleus.
Studies
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Study Purpose
This study evaluated how circulating tumor cell (CTC) counts are influenced by a pre-processing delay and inter-laboratory, intra-assay, inter-instrument, and inter-operator variability when enumerated using the CellSearch system. Peripheral blood was collected from six metastatic cancer patients and an unspecified number of healthy volunteers. The first 3 mL of blood collected from each individual was discarded to prevent epithelial cell contamination and 120 mL of blood was collected from each individual in CellSave tubes, pooled, and then realiquoted into new CellSave tubes (8.5 mL/tube). Inter-laboratory results were assessed by transporting three specimen aliquots by courier (two cohorts) to 13 or 14 participating laboratories for analysis using the CellSearch Epithelial Cell Kit and the CellTracks AutoPrep System with a semi-automated fluorescence microscope. Specimen CTC scores were based on the clinically relevant cut-off levels of ≥3 CTC/7.5 ml for metastatic colon carcinoma and ≥5 CTC/7.5 ml blood for other advanced carcinomas. Potential effects on CTC enumeration were evaluated by comparing results obtained from specimens processed within 1, 2, 3, or 5 days post-collection. Potential effects of a post-processing delay were evaluated using aliquots obtained from a single metastatic cancer patient by storing blood after initial analysis in the dark for 24 h before re-analysis. Inter-instrument variation was assessed by including high and low control specimens of the same batch in each cohort. Intra-assay variability was assessed by comparing results obtained when five aliquots from a single pooled blood specimen were analyzed as a single batch on one instrument by the coordinating center. To assess inter-operator variability, a total of 509 blinded images representing blood specimens collected from each of the six metastatic cancer patients were provided to participating centers by the coordinating center as on online survey to gauge variability associated with interpretation.
Summary of Findings:
Of the six specimens collected from metastatic cancer patients, three specimens were correctly classified by all 14 laboratories based on clinically relevant cut-off levels of ≥3 or 5 CTC/7.5 ml of blood, but discordant results were reported for the remaining three specimens at 7%, 15%, and 29% of the testing centers, respectively. The inter-laboratory coefficient of variation (CV) for each of the six specimens ranged between 45-64%, with an overall discordance of 7.5% for the 80 aliquots analyzed. Participating laboratories reported similar median CTC counts (2-9 CTC/7.5 ml blood) among the 77 blood aliquots that were analyzed within 1 day (27 aliquots), 2 days (43 aliquots), or 3 days (7 aliquots) after collection; however, median CTC count was higher (16 CTC/7.5 ml blood) among the 3 aliquots stored for 5 days prior to processing. Notably, a wide range in CTC counts was also observed among all aliquots regardless of a processing delay (1 d: 1-65 CTC, 2 d: 0-55 CTC, 3 d: 1-23 CTC, 5 d: 11-19 CTC). Inter-instrument variation was determined to be low (CV<12%) based on median CTC counts obtained with high- and low-level controls. Inter-assay variation was very low for both specimens tested using five replicate assays (CV=20% and CV=14%, respectively). Initial results were also consistent with those obtained after a specimen was stored for 24 h in the dark post-analysis. Inter-operator agreement was classified as "substantial" for four blood specimens and "almost perfect" for the remaining two specimens based on Fleiss' K statistics; however, discordance in image interpretation was present and ranged between 4 and 31% of the 509 images evaluated. The authors specified that scoring inconsistencies among laboratories was most prominent among samples that contained high numbers of cells that were dead, apoptotic, or lacked a nucleus.
Biospecimens
Preservative Types
- Other Preservative
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Cell count/volume Fluorescent microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Time at room temperature 1 day
2 days
3 days
5 days
Analyte Extraction and Purification Between site variability Multiple instruments compared
Multiple processing centers compared